Live-cell imaging unveils distinct R-loop populations with heterogeneous dynamics

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• R-loops, which are RNA:DNA hybrids, form during transcription and are implicated in genome stability and disease.
• Existing methods for studying R-loops often lack the specificity or real-time imaging capabilities needed for dynamic cellular studies.

Purpose of the Study:

• To develop a novel, genetically encoded sensor for live-cell imaging of R-loops.
• To characterize the abundance, dynamics, and regional variations of R-loops in live cells.

Main Methods:

• Development of RHINO, a sensor comprising RNA:DNA hybrid binding domains of human RNase H1 fused to a fluorescent protein.
• Utilizing RHINO for high-specificity and sensitivity measurement of R-loop dynamics in live cells.
• Kinetic analysis of R-loops at nucleoli, telomeres, and protein-coding genes.

Main Results:

• RHINO enables precise live-cell imaging of R-loops with high specificity and sensitivity.
• Significant variations in R-loop dynamics were observed across nucleoli, telomeres, and protein-coding genes.
• A kinetic framework for R-loop behavior in different genomic contexts was established.

Conclusions:

• RHINO is a powerful tool for studying R-loop biology in real-time within live cells.
• R-loop dynamics are context-dependent, suggesting specialized roles in various chromosomal regions.
• Understanding R-loop dynamics is crucial for elucidating their contribution to cellular processes and disease pathogenesis.